Blue Native PAGE of PINK1-TOM complex in mammalian cells v1

Autosomal recessive mutations in PTEN-induced kinase 1 (PINK1) are associated with early-onset Parkinson's disease (PD) [1]. Upon inducing mitochondrial depolarization using uncouplers, the full-length human PINK1 stabilizes at mitochondria with the TOM (translocase of the outer mitochondrial membrane) complex, undergoing autophosphorylation and activation [2]. PINK1 plays a crucial role in phosphorylating ubiquitin and the E3 ubiquitin ligase, Parkin, initiating mitophagy for selectively removing damaged mitochondria [3]. To investigate PINK1 mutation impacts on PINK1-TOM complex formation, Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) was done on stable cell lines in PINK1 knockout (KO) HeLa cells expressing doxycycline-induced PINK1/mutants. BN-PAGE substitutes SDS with Coomassie G250, preserving proteins in their native conformation, and utilizes non-ionic detergents for solubilization, ensuring complex integrity during analysis [4, 5]. Studying the PINK1-TOM complex has proved vital for our understanding of PINK1 stabilization on mitochondria. Here, we share our method for resolving protein complexes by blue native PAGE for PINK1-TOM complexes.